This invention is directed to synthetic procedures for simultaneous crosslinking and functionalization of polysaccharide chromatography media.
Crosslinked polysaccharide media have become very important for chromatographic separations during the past decades. The most important areas for the use of crosslinked polysaccharide media are the separation of biomolecules such as proteins, nucleic acids, and carbohydrates.
Polysaccharide chromatography media are typically macroporous and the porosity is a function of the concentration of polysaccharide used to produce these gel matrices. Typically, the lower the carbohydrate concentration, the bigger the pores are, i.e., the higher the exclusion limit. The size of the pores is critical for the separation. The porous polysaccharide gels are further derivatized with various functionalities such as ion exchange, hydrophobic interaction, or affinity functionalities to effect separation of biomolecules.
The polysaccharide gel matrices are composed of a great number of intertwining polymeric chains that are held together by hydrogen bonding. The polysaccharide chains attain certain fixed distances and form chain-like structures between which the pores are formed. The crosslinking within these structures does not significantly affect the size of the pores, only the rigidity of the particle. The crosslinking of these chains utilizes hydroxyl groups on the polysaccharides and leads to chemically stable particles. Several methods have been published in connection with cellulose (British Patent No. 1,026,706), dextran (U.S. Pat. Nos. 3,042,677 and 3,208,994) and agarose crosslinked with epichlorohydrin (U.S. Pat. No. 3,507,851) and other bifunctional reagents (U.S. Pat. No. 3,860,573). Other bifunctional reagents utilized are divinyl sulfone, bisepoxides, and dicarboxylic acid chlorides (British Patent No. 1,352,613).
Lindgren (U.S. Pat. No. 4,973,683) described the mechanism of conventional crosslinking methods referring to the work of L. Holmberg (Doctoral Thesis, Swedish University of Agricultural Sciences 1983. pp. 28-29). Holmberg has demonstrated that upon crosslinking of polysaccharides with epichlorohydrin the primary mechanism of crosslinking is polymer crosslinking and substitution and very little monomer crosslinking takes place in which only monomer epichlorohydrin participates. This explains why the rigidity of the gel matrix does not increase significantly by this type of crosslinking. To eliminate polymerization of the crosslinker, Lindgren suggested that bifunctional crosslinkers with functional groups that can be activated under a controlled reaction sequence are optimal for increasing the rigidity of agarose gels. One functional group is used to introduce the crosslinker into the agarose matrix via the available hydroxyl groups. The second functional group remains unreactive under the conditions employed to introduce the crosslinker. Then the second functional group of the crosslinker is activated and brought into reaction with an adjacent hydroxyl group thus forming a crosslink between the polysaccharide chains. The repetition of these steps leads to increasing number of crosslinks and increased rigidity of the particle. This is a controlled procedure during which side-reactions are excluded and optimal crosslinking is obtained. When the required rigidity is attained, the particle is suitable to be functionalized with the required groups (affinity, ion exchange, hydrophobic interaction, etc.) to prepare the desired type chromatography gel.
Although the methods of Lindgren are useful, they have the drawback that crosslinking and functionalization occur in successive steps. This increases the time required, reduces the overall yield, and increases the possibility of side reactions and other loss of material. Therefore, there is a need for an improved method that can allow simultaneous crosslinking and functionalization of polysaccharide chromatography media.